Search results for "Density gradient"

The purification and properties of nucleoside phosphotransferase from mucosa of chicken intestine

1984

Abstract (1) Nucleoside phosphotransferase (nucleotide:3′-deoxynucleoside 5′-phosphotransferase, EC 2.7.1.77) has been purified from chicken intestine mucosa to apparent homogeneity. The enzyme is represented by a multisubunit protein at different degrees of association. It can dissociate into a compoenent with a marked fall in catalytic activity. (2) The associated forms are similar to the enzyme previously purified from chick embryo as regards: substrate specificity both with respect to nucleoside monophosphate donors and to deoxyribonucleoside acceptors; sigmoidicity in the rate curve with a variable phosphate donor; instability to heat, dilution and lowering of pH; the activating and pr…

Studies on sea urchin oocytes. II. Synthesis of RNA during oogenesis.

1972

Abstract Isolated oocytes of the sea urchin Paracentrotus lividus actively incorporate 3H-uridine into RNA. Labeled RNA was analysed by sucrose gradient and acrylamide gel electrophoresis following cell fractionation. Much of the radioactivity is incorporated at the nucleolar level in the form of rRNA precursors. The kinetics of maturation of these latter suggests that this occurs at a slower rate than during embryogenesis. Other non-nucleolar RNA classes are also actively labelled and retained in the nucleus for many hours. These results are confirmed by an autoradiographic investigation.

Echovirus 1 Endocytosis into Caveosomes Requires Lipid Rafts, Dynamin II, and Signaling EventsV⃞

2004

Binding of echovirus 1 (EV1, a nonenveloped RNA virus) to the α2β1 integrin on the cell surface is followed by endocytic internalization of the virus together with the receptor. Here, video-enhanced live microscopy revealed the rapid uptake of fluorescently labeled EV1 into mobile, intracellular structures, positive for green fluorescent protein-tagged caveolin-1. Partial colocalization of EV1 with SV40 (SV40) and cholera toxin, known to traffic via caveosomes, demonstrated that the vesicles were caveosomes. The initiation of EV1 infection was dependent on dynamin II, cholesterol, and protein phosphorylation events. Brefeldin A, a drug that prevents SV40 transport, blocked the EV1 infection…

Chromospheric evaporation and phase mixing of Alfvén waves in coronal loops

2020

Phase mixing of Alfv\'en waves has been studied extensively as a possible coronal heating mechanism but without the full thermodynamic consequences considered self-consistently. It has been argued that in some cases, the thermodynamic feedback of the heating could substantially affect the transverse density gradient and even inhibit the phase mixing process. In this paper, we use MHD simulations with the appropriate thermodynamical terms included to quantify the evaporation following heating by phase mixing of Alfv\'en waves in a coronal loop and the effect of this evaporation on the transverse density profile. The numerical simulations were performed using the Lare2D code. We set up a 2D l…

Thirty years of synaptosome research.

1993

Detached synapses (synaptosomes), first isolated by the author in 1958 and identified as such in 1960, are sealed presynaptic nerve terminals often with a portion of the target cell--sometimes amounting to a complete dendritic spine--adhering to their external surface. They can be prepared in high yield from brain tissue and also in decreasing yield from spinal cord, retina, sympathetic ganglia, myenteric plexus and electric organs. They are sealed structures which, under metabolizing conditions, respire, take up oxygen and glucose, extrude Na+, accumulate K+, maintain a normal membrane potential and, on depolarization, release transmitter in a Ca(2+)-dependent manner. They thus provide an …

A homemade device for linear sucrose gradients

2008

We have developed a simple and inexpensive device to obtain linear sucrose gradients with commonly used laboratory materials--a syringe, a flask, a plastic tube, and a piece of Pongo (Play-Doh). Refractive index values measured on sucrose fractions collected using our system demonstrate both the linearity and reliability of the gradients obtained.

Assays of Proteasome-Dependent Cleavage Products

2005

The degradation of misfolded, aged, or no longer needed cytosolic proteins depends largely on the ubiquitin-proteasome system. Proteasomes degrade their substrates into fragments of 3-20 amino acids. Human 20S proteasomes can be purified from human erythrocytes by batch adsorption to DEAE-cellulose, ammonium sulfate precipitation, anion-exchange fast protein liquid chromatography (FPLC), and glycerol density gradient ultracentrifugation. 20S proteasomes purified by this method are suitable for the in vitro digestion of synthetic peptides as well as full-length proteins. The degradation products produced by proteasomes are separated by reversed-phase HPLC using an acetonitrile gradient. The …

1990

The determination of average molecular weights and the characterization of molecular weight distributions by the ratio of the weight- to number-average molecular weights Mw/Mn from sedimentation equilibrium in the ultracentrifuge are described. The essential measurement of the density profile of the mixed solvent is conducted according to Munk (Macromolecules15, 500 (1982)). Special evaluation of the polymer band in the equilibrium allowed to deduce from the observed concentration profile Mw and Mn of a series of poly(methyl methacrylate) samples and thus to characterize the molecular weight distribution of the samples.

Effect of digitonin on membrane-bound and chitosomal chitin synthetase activity in protoplasts from yeast cells ofCandida albicans

1993

The effect of digitonin on chitin synthetase present in membrane (MMF) and cytoplasmic fractions (chitosomes) (CF) from C. albicans yeast protoplasts has been determined. The zymogen is preferentially, but not exclusively, solubilized by digitonin from MMF. Centrifugation of distinct solubilized preparations, containing either zymogen, in vivo active enzyme and/or trypsin activated enzyme, on linear sucrose gradients suggests that both zymogen and trypsin activated enzyme sediment slightly slower than the active enzyme, pointing out differences between the activation processes in vivo and in vitro or, alternatively, that both enzyme activities (active in vivo and zymogenic) correspond to di…

Isolation of nuclei and downstream processing of cell-type-specific nuclei from micro-dissected mouse brain regions – techniques and caveats

2020

AbstractThe mammalian brain consists of several structurally and functionally distinct regions equipped with an equally complex cell-type system. Due to its relevance in uncovering disease mechanisms, the study of cell-type-specific molecular signatures of different brain regions has increased. The rapid evolution of newer and cheaper sequencing techniques has also boosted the interest in cell-type-specific epigenetic studies. In fact, the nucleus holds most of the cell’s epigenetic information and is quite resistant to tissue dissociation processes as compared to cells. As such, nuclei are continually preferred over cells for epigenetic studies. However, the isolation of nuclei from cells …